As used herein, citations to references are indicated in brackets, and are further described in the “References Cited” listing contained herein.
Enzymes that catalyze biosynthesis of steroid hormones in human are mainly cytochrome P450s and non-metallo dehydrogenases/reductases [A1]. Cytochrome P450's are members of a superfamily of heme-containing enzymes present both in eukaryotes and prokaryotes [A2]. Human cytochrome P450's have 18 gene families and 44 subfamilies. Cytochrome P450 Aromatase is the product of the CYP19A1 gene on chromosome 15q21.1, which has one family and one subfamily. The class I cytochrome P450s are mitochondrial and receive electrons via an iron-sulfur protein adrenodoxin and a flavoprotein adrenodoxin reductase. Class II enzymes, on the other hand, are residents of the endoplasmic reticulum/golgi system and use the flavoprotein cytochrome P450 reductase (CPR) to receive electrons from NADPH. Of the 57 human sequenced cytochrome P450 genes, 7 belong to Class I, and 50 to class II [A3,4].
Cytochrome P450 Aromatase (henceforth Aromatase) is one of the most important class II cytochrome P450s involved in steroid biosynthesis. Aromatase uses with high specificity androstenedione, testosterone, and 16α-hydroxytestosterone (all with the same androgen backbone) as substrates converting them to estrone, 17β-estradiol, and 17β,16α-estriol (all with the same estrogen backbone), respectively. It is the only known enzyme in vertebrates capable of catalyzing the aromatization of a six-membered ring. The functional human enzyme is monomeric, comprised of a heme group and a single polypeptide chain of 503 amino-acid residues (molecular mass about 55 kDa). It is an integral membrane protein of the endoplasmic reticulum, anchored to the membrane by an amino terminal transmembrane domain [A5-7], in addition to other membrane-associating regions.
Many soluble bacterial cytochrome P450s including P450cam [A8], P450BM-3 [A9], P450terp [A10], and P450eryF [A11], have been crystallized and structures determined by X-ray crystallography. In recent times, crystal structures of several recombinant, microsomal human cytochrome P450s (PDB ID codes: 1A2, 2A6, 2A13, 2C8, 2C9, 2D6, 2R1 and 3A4) have been determined [A12-16 and references therein]. Nearly all of these P450s catalyze metabolism of a wide variety of endogenous and xenobiotic compounds and drugs with low substrate specificities.
Being the sole catalyst for a unique hydroxylation, carbon-carbon bond cleavage and ring aromatization reaction step in the estrogen biosynthesis pathway, Aromatase has been the subject of intense biochemical and biophysical investigations for the past 35 years [see A7, 17, 18 for reviews]. Nevertheless, many aspects of the Aromatase catalyzed reaction, especially the third aromatization step, remain poorly understood. Lack of a crystal structure of Aromatase has led to a number of homology models for the enzyme based on other experimental P450 structures and site-directed mutagenesis data [A19-30 and references therein]. Several androgen-binding scenarios at the active site, possible involvements of side chains in the catalytic process, as well as models for enzyme's mechanism of action have been proposed based on these structural and functional analyses [A20-24, 27-29]. However, validation of all these results necessitated an experimental three-dimensional model of the enzyme showing the binding mode of the steroidal substrate and its interactions with active side amino acids. Additionally, because inhibition of estrogen biosynthesis by Aromatase inhibitors (AI) constitutes one of the foremost therapies for postmenopausal estrogen-dependent breast cancer today [A30-32], details of the substrates and inhibitor binding interactions at the active site have become increasingly critical information for the development of next generation AIs.
Despite concerted efforts in many laboratories, no experimental molecular structure of Aromatase has emerged yet. The major impediments to Aromatase crystallization have been its strong hydrophobic character, and susceptibility to rapid denaturation in the absence of the protective lipid bilayer. Furthermore, recombinant DNA techniques have also thus far been unsuccessful in producing the enzyme in qualities and quantities suitable for crystallization. A number of laboratories have reported purification of aromatase from human placenta [A33-35] and recombinant expression systems [A30, 36]. Nevertheless, attempts to crystallize either the placental or a recombinant/modified aromatase have been unsuccessful and an experimental aromatase structure has remained elusive. Numerous mechanistic/homology models based on known P450 structures and site-directed mutagenesis data have been proposed [A19-30], none of which could satisfactorily explain the functional data or enzyme action. Using term human placenta as a rich source of Aromatase and an elegant purification technique that employs a highly specific monoclonal antibody-based affinity chromatography [A37], we have been able to purify large quantities of the enzyme in a pristine, active form that has permitted the growth of diffraction-quality single crystals under suitable detergent conditions.
Therefore, there is a need for a purification and crystallization procedure that can yield a crystal of at least one binding site of a human aromatase, thereby providing a crystal and associated data and information that can be used to design and screen for drugs and new compounds for treating androgen-dependent breast cancer and for modulating estrogen biosynthesis. The present invention is useful in addressing this and other needs.